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1.
Clin Transl Allergy ; 11(4): e12035, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34161665

ABSTRACT

BACKGROUND: Berotralstat (BCX7353) is an oral, once-daily inhibitor of plasma kallikrein recently approved for prevention of angioedema attacks in adults and adolescents with hereditary angioedema (HAE). The objective of this report is to summarize results from an interim analysis of an ongoing long-term safety study of berotralstat in patients with HAE. METHODS: APeX-S is an ongoing, phase 2, open-label study conducted in 22 countries (ClinicalTrials.gov, NCT03472040). Eligible patients with a clinical diagnosis of HAE due to C1 inhibitor deficiency (HAE-C1-INH) were centrally allocated to receive berotralstat 150 or 110 mg once daily. The primary objective was to determine long-term safety and the secondary objective was to evaluate effectiveness. RESULTS: Enrolled patients (N = 227) received berotralstat 150 mg (n = 127) or 110 mg (n = 100) once daily. The median (range) duration of exposure was 342 (11-540) and 307 (14-429) days for the 150-mg and 110-mg groups, respectively. Treatment-emergent adverse events (TEAEs) occurred in 91% (n = 206) of patients. The most common TEAEs across treatment groups were upper respiratory tract infection (n = 91, 40%), abdominal pain (n = 57, 25%), headache (n = 40, 18%), and diarrhea (n = 31, 14%) and were mostly mild to moderate. Fifty percent (n = 113) of patients had at least one drug-related adverse event (AE; 150 mg, n = 57 [45%]; 110 mg, n = 56 [56%]), and discontinuations due to AEs occurred in 19 (8%) patients (150 mg, n = 13 [10%]; 110 mg, n = 6 [6%]). Three (1.3%) patients experienced a drug-related serious TEAE. Among patients who received berotralstat through 48 weeks (150 mg, n = 73; 110 mg, n = 30), median HAE attack rates were low in month 1 (150 mg, 1.0 attacks/month; 110 mg, 0.5 attacks/month) and remained low through 12 months (0 attacks/month in both dose groups). Mean HAE attack rates followed a similar trend, and no evidence for patient tolerance to berotralstat emerged. In both dose groups, angioedema quality of life scores showed clinically meaningful changes from baseline. CONCLUSIONS: In this analysis, both berotralstat doses, 150  and 110 mg once daily, were generally well tolerated. Effectiveness results support the durability and robustness of berotralstat as prophylactic therapy in patients with HAE. TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov (NCT03472040).

2.
J Allergy Clin Immunol Pract ; 9(6): 2305-2314.e4, 2021 06.
Article in English | MEDLINE | ID: mdl-33866032

ABSTRACT

BACKGROUND: Berotralstat (BCX7353) is a recently approved, oral, once-daily kallikrein inhibitor for hereditary angioedema (HAE) prophylaxis. In the APeX-2 trial, berotralstat reduced HAE attack rates over 24 weeks, with a favorable safety and tolerability profile. OBJECTIVE: Evaluate berotralstat safety, tolerability, and effectiveness over 48 weeks. METHODS: APeX-2 is a phase 3, parallel-group, multicenter trial (NCT03485911) in patients with HAE due to C1 esterase inhibitor deficiency. Part 1 was double-blind and placebo-controlled, with patients randomized to 24 weeks of berotralstat 150 mg, 110 mg, or placebo. In part 2, patients continued berotralstat the same dose or, if initially randomized to placebo, were rerandomized to berotralstat 150 mg or 110 mg through weeks 24 to 48. The primary end point was safety and tolerability. RESULTS: One hundred eight patients received 1 or more doses of berotralstat in part 2. Treatment-emergent adverse events (TEAEs) occurred in 30 of 39 patients (77%) in the placebo group during part 1, and 25 of 34 patients (74%) re-randomized from placebo to berotralstat 110 mg or 150 mg in part 2, with drug-related TEAEs in 13 of 39 (33%), and 11 of 34 (32%) in the same groups. Most TEAEs were mild or moderate, with no serious drug-related TEAEs. The most common TEAEs were upper respiratory tract infections, abdominal pain, diarrhea, and vomiting. Mean (±standard error of the mean) monthly attack rates at baseline and week 48 were 3.06 (±0.25) and 1.06 (±0.25) in the berotralstat 150mg 48-week group and 2.97 (±0.21) and 1.35 (±0.33) in the berotralstat 110mg 48-week group. CONCLUSIONS: The safety, tolerability, and effectiveness of berotralstat were maintained over 48 weeks of treatment.


Subject(s)
Angioedemas, Hereditary , Angioedemas, Hereditary/drug therapy , Double-Blind Method , Humans , Pyrazoles , Treatment Outcome
3.
Allergy ; 76(6): 1789-1799, 2021 06.
Article in English | MEDLINE | ID: mdl-33247955

ABSTRACT

BACKGROUND: With no approved treatments in Japan for the prevention of hereditary angioedema (HAE) attacks, there is a significant unmet need for long-term prophylactic therapies for Japanese patients with HAE. Berotralstat (BCX7353) is an oral, once-daily, highly selective inhibitor of plasma kallikrein in development for prophylaxis of angioedema attacks in HAE patients. METHODS: APeX-J is a phase 3, randomized, double-blind, placebo-controlled, parallel-group, 3-part trial conducted in Japan (University Hospital Medical Information Network identifier, UMIN000034869; ClinicalTrials.gov identifier, NCT03873116). Patients with a clinical diagnosis of type 1 or 2 HAE underwent a prospective run-in period of 56 days to determine eligibility, allowing enrollment of those with ≥2 expert-confirmed angioedema attacks. Patients were randomly assigned (1:1:1) and stratified by baseline attack rate (≥2 vs. <2 expert-confirmed attacks/month between screening and randomization) to receive once-daily berotralstat 110 mg, berotralstat 150 mg, or placebo. The primary endpoint was the rate of expert-confirmed angioedema attacks during dosing in the 24-week treatment period. RESULTS: Nineteen patients were randomized to receive once-daily berotralstat 110 mg (n = 6), berotralstat 150 mg (n = 7), or placebo (n = 6). Treatment with berotralstat 150 mg significantly reduced HAE attacks relative to placebo (1.11 vs. 2.18 attacks/month, p = .003). The most frequently reported treatment-emergent adverse events (TEAEs) in berotralstat-treated patients (n = 13) were nasopharyngitis (n = 4, 31%), abdominal pain, cough, diarrhea, and pyrexia (n = 2 each, 15%). CONCLUSIONS: Orally administered, once-daily berotralstat 150 mg significantly reduced the frequency of HAE attacks and was safe and well tolerated, supporting its use as a prophylactic therapy in patients with type 1 or 2 HAE in Japan.


Subject(s)
Angioedemas, Hereditary , Angioedemas, Hereditary/diagnosis , Angioedemas, Hereditary/drug therapy , Angioedemas, Hereditary/epidemiology , Complement C1 Inhibitor Protein/adverse effects , Humans , Japan/epidemiology , Prospective Studies , Pyrazoles
4.
J Allergy Clin Immunol ; 148(1): 164-172.e9, 2021 07.
Article in English | MEDLINE | ID: mdl-33098856

ABSTRACT

BACKGROUND: Berotralstat (BCX7353) is an oral, once-daily inhibitor of plasma kallikrein in development for the prophylaxis of hereditary angioedema (HAE) attacks. OBJECTIVE: Our aim was to determine the efficacy, safety, and tolerability of berotralstat in patients with HAE over a 24-week treatment period (the phase 3 APeX-2 trial). METHODS: APeX-2 was a double-blind, parallel-group study that randomized patients at 40 sites in 11 countries 1:1:1 to receive once-daily berotralstat in a dose of 110 mg or 150 mg or placebo (Clinicaltrials.gov identifier NCT03485911). Patients aged 12 years or older with HAE due to C1 inhibitor deficiency and at least 2 investigator-confirmed HAE attacks in the first 56 days of a prospective run-in period were eligible. The primary efficacy end point was the rate of investigator-confirmed HAE attacks during the 24-week treatment period. RESULTS: A total of 121 patients were randomized; 120 of them received at least 1 dose of the study drug (n = 41, 40, and 39 in the 110-mg dose of berotralstat, 150-mg of dose berotralstat, and placebo groups, respectively). Berotralstat demonstrated a significant reduction in attack rate at both 110 mg (1.65 attacks per month; P = .024) and 150 mg (1.31 attacks per month; P < .001) relative to placebo (2.35 attacks per month). The most frequent treatment-emergent adverse events that occurred more with berotralstat than with placebo were abdominal pain, vomiting, diarrhea, and back pain. No drug-related serious treatment-emergent adverse events occurred. CONCLUSION: Both the 110-mg and 150-mg doses of berotralstat reduced HAE attack rates compared with placebo and were safe and generally well tolerated. The most favorable benefit-to-risk profile was observed at a dose of 150 mg per day.


Subject(s)
Angioedemas, Hereditary/drug therapy , Pyrazoles/administration & dosage , Administration, Oral , Adult , Double-Blind Method , Female , Humans , Male , Plasma Kallikrein/administration & dosage , Prospective Studies , Treatment Outcome
5.
J Neurosci ; 30(47): 15811-20, 2010 Nov 24.
Article in English | MEDLINE | ID: mdl-21106820

ABSTRACT

Inflammation is increasingly recognized as an important contributor to a host of CNS disorders; however, its regulation in the brain is not well delineated. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the inflammasome complex, which also includes ASC (apoptotic speck-containing protein with a card) and procaspase-1. Inflammasome formation can be triggered by membrane P2X(7)R engagement leading to cleavage-induced maturation of caspase-1 and interleukin-1ß (IL-1ß)/IL-18. This work shows that expression of the Nlrp3 gene was increased >100-fold in a cuprizone-induced demyelination and neuroinflammation model. Mice lacking the Nlrp3 gene (Nlrp3(-/-)) exhibited delayed neuroinflammation, demyelination, and oligodendrocyte loss in this model. These mice also showed reduced demyelination in the experimental autoimmune encephalomyelitis model of neuroinflammation. This outcome is also observed for casp1(-/-) and IL-18(-/-) mice, whereas IL-1ß(-/-) mice were indistinguishable from wild-type controls, indicating that Nlrp3-mediated function is through caspase-1 and IL-18. Additional analyses revealed that, unlike the IL-1ß(-/-) mice, which have been previously shown to show delayed remyelination, Nlrp3(-/-) mice did not exhibit delayed remyelination. Interestingly, IL-18(-/-) mice showed enhanced remyelination, thus providing a possible compensatory mechanism for the lack of a remyelination defect in Nlrp3(-/-) mice. These results suggest that NLRP3 plays an important role in a model of multiple sclerosis by exacerbating CNS inflammation, and this is partly mediated by caspase-1 and IL-18. Additionally, the therapeutic inhibition of IL-18 might decrease demyelination but enhance remyelination, which has broad implications for demyelinating diseases.


Subject(s)
Carrier Proteins/physiology , Caspase 1/physiology , Demyelinating Diseases/pathology , Encephalomyelitis, Autoimmune, Experimental/pathology , Inflammasomes/physiology , Inflammation Mediators/physiology , Interleukin-18/physiology , Animals , Carrier Proteins/genetics , Caspase 1/deficiency , Caspase 1/genetics , Demyelinating Diseases/enzymology , Demyelinating Diseases/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Inflammasomes/deficiency , Inflammasomes/genetics , Inflammation Mediators/metabolism , Interleukin-18/deficiency , Interleukin-18/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiple Sclerosis/enzymology , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , NLR Family, Pyrin Domain-Containing 3 Protein , Signal Transduction/genetics , Signal Transduction/physiology
6.
J Immunol ; 185(2): 974-81, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20574004

ABSTRACT

The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro-IL-1beta and pro-IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3(-/-) mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8(+) T cells in the spinal cords of the Nlrp3(-/-) mice, whereas microglial accumulation remained the same. Nlrp3(-/-) mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3(-/-) mice with EAE produced less IL-18, and the disease course was similar to Il18(-/-) mice. Furthermore, Nlrp3(-/-) and Il18(-/-) mice had similarly reduced IFN-gamma and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1-dependent cytokines which then influence Th1 and Th17.


Subject(s)
Carrier Proteins/physiology , Encephalomyelitis, Autoimmune, Experimental/immunology , Multiple Sclerosis/immunology , T-Lymphocytes, Helper-Inducer/immunology , Th1 Cells/immunology , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 1/genetics , Caspase 1/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Gliosis/immunology , Gliosis/metabolism , Gliosis/pathology , Humans , Immunoblotting , Interferon-gamma/metabolism , Interleukin-17/metabolism , Interleukin-18/genetics , Interleukin-18/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiple Sclerosis/pathology , Multiple Sclerosis/physiopathology , Myelin Sheath/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Severity of Illness Index , Spinal Cord/immunology , Spinal Cord/metabolism , Spinal Cord/pathology , T-Lymphocytes, Helper-Inducer/metabolism , Th1 Cells/metabolism
7.
Proc Natl Acad Sci U S A ; 105(35): 13015-20, 2008 Sep 02.
Article in English | MEDLINE | ID: mdl-18728195

ABSTRACT

The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell-cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor-ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Immunity/immunology , Semaphorins/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , Cell Proliferation , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Semaphorins/antagonists & inhibitors , Signal Transduction
8.
J Neuroimmunol ; 194(1-2): 97-106, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18207576

ABSTRACT

Inflammatory cytokines have been implicated in the pathology of multiple neurologic diseases, including multiple sclerosis. We examined the role of the TNF family member TWEAK in neuroinflammation. Cuprizone-fed mice undergo neuroinflammation and demyelination in the brain, but upon removal of cuprizone from the diet, inflammation is resolved and remyelination occurs. Using this model, we demonstrate that mice lacking TWEAK exhibit a significant delay in demyelination and microglial infiltration. During remyelination, mice lacking the TWEAK gene demonstrate only a marginal delay in remyelination. Thus, this study identifies a primary role of TWEAK in promoting neuroinflammation and exacerbating demyelination during cuprizone-induced damage.


Subject(s)
Demyelinating Diseases/physiopathology , Encephalitis/physiopathology , Tumor Necrosis Factors/physiology , Animals , Astrocytes/metabolism , Astrocytes/pathology , Chelating Agents/toxicity , Copper , Cuprizone/toxicity , Cytokine TWEAK , Demyelinating Diseases/chemically induced , Double-Blind Method , Encephalitis/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/metabolism , Microglia/pathology , Myelin Sheath/physiology , Oligodendroglia/metabolism , Oligodendroglia/pathology , RNA, Messenger/biosynthesis , Receptors, Tumor Necrosis Factor/physiology , TWEAK Receptor , Tumor Necrosis Factors/biosynthesis , Tumor Necrosis Factors/deficiency , Tumor Necrosis Factors/genetics
9.
J Neurosci ; 27(28): 7429-37, 2007 Jul 11.
Article in English | MEDLINE | ID: mdl-17626203

ABSTRACT

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.


Subject(s)
Demyelinating Diseases/physiopathology , Lymphotoxin beta Receptor/metabolism , Animals , Apoptosis , Brain/metabolism , Brain/pathology , Cell Division , Corpus Callosum/pathology , Cuprizone/pharmacology , Demyelinating Diseases/chemically induced , Demyelinating Diseases/metabolism , Demyelinating Diseases/pathology , Immunoglobulin G/genetics , Lymphotoxin beta Receptor/antagonists & inhibitors , Lymphotoxin beta Receptor/deficiency , Lymphotoxin beta Receptor/genetics , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/pathology , Myelin Sheath/drug effects , Myelin Sheath/ultrastructure , Oligodendroglia , Recombinant Fusion Proteins/pharmacology , Time Factors , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism
10.
J Immunol ; 177(7): 4252-6, 2006 Oct 01.
Article in English | MEDLINE | ID: mdl-16982856

ABSTRACT

Porphyromonas gingivalis (Pg) is a major etiologic agent for chronic periodontitis. Tissue destruction by Pg results partly from induction of host inflammatory responses through TLR2 signaling. This work examines the role of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), an adaptor molecule important for TLR-mediated caspase-1 activation. Results demonstrate that ASC levels are stable upon infection of human THP1 monocytic cells with Pg but decrease after cytokine induction. Using short hairpin RNA, we demonstrate an essential role for ASC in induction of IL-1beta by TLR2, 4, and 5 agonists, live Escherichia coli, and Pg. Induction of IL-6, IL-8, IL-10, and TNF also requires ASC, but this induction is not inhibited by IL-1 receptor antagonist or caspase-1 inhibitor. Similar results in U937 indicate broad applicability of these findings. Pg-infected ASC knockdown THP1 cells exhibit reduced transcript levels and NF-kappaB activation. These results suggest a role for ASC in cytokine induction by Pg involving both caspase-1-dependent and -independent mechanisms.


Subject(s)
Bacteroidaceae Infections/immunology , Caspase 1/metabolism , Cytokines/biosynthesis , Cytoskeletal Proteins/immunology , Porphyromonas gingivalis/immunology , Blotting, Western , CARD Signaling Adaptor Proteins , Cytoskeletal Proteins/genetics , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Escherichia coli/immunology , Humans , Interleukin-1/biosynthesis , Interleukin-10/biosynthesis , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , NF-kappa B/metabolism , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 2/agonists , Toll-Like Receptor 4/agonists , Toll-Like Receptor 5/agonists , Tumor Necrosis Factor-alpha/biosynthesis , U937 Cells
11.
BMC Biotechnol ; 6: 7, 2006 Jan 24.
Article in English | MEDLINE | ID: mdl-16433925

ABSTRACT

BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.


Subject(s)
Algorithms , Gene Silencing , Gene Targeting/methods , RNA Probes/genetics , RNA, Small Interfering/genetics , Sequence Analysis, RNA/methods , Drug Design , Genetic Vectors/genetics , Sequence Alignment/methods
12.
Am J Pathol ; 163(2): 465-76, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12875968

ABSTRACT

The cytokine tumor necrosis factor (TNF)-alpha has previously been shown to prime hepatocytes to a state of replicative competence, but has not been shown to act as a complete mitogen for these cells. In the present study we have altered our previously described long-term dimethyl sulfoxide culture system to exclude all known hepatocyte mitogens from the culture media and enable us to directly examine the effects of TNF-alpha on primary rat hepatocytes. We have shown that cells maintained under these culture conditions retain the biochemical and morphological features of well-differentiated hepatocytes. Treatment with TNF-alpha induced DNA synthesis relative to control, to a level not significantly different from that induced by the known hepatocyte mitogen, epidermal growth factor (EGF). Maximal DNA synthesis was induced by treatment with 250 U/ml TNF-alpha for 24 hours. Mitotic figures were observed in cultures treated with TNF-alpha or EGF but not in untreated controls. Treatment of cultures with TNF-alpha, but not EGF, induced activation of both nuclear factor-kappaB p50 homodimers and p50/p65 heterodimers. DNA synthesis induced by TNF-alpha was inhibited by treatment with transforming growth factor-beta. Based on the results of our studies, we conclude that TNF-alpha acts as a complete mitogen for rat hepatocytes.


Subject(s)
Hepatocytes/drug effects , Mitogens/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Biomarkers , Cell Size , Cells, Cultured , DNA/metabolism , Epidermal Growth Factor/metabolism , Hepatocytes/physiology , Hepatocytes/ultrastructure , Humans , Male , Mice , NF-kappa B/metabolism , Rats , Rats, Inbred F344 , Time Factors
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